Despite increased plasma concentrations of HDL, as well as apoA-I, LCAT transgenic (L-tg) mice have enhanced (up to 5-fold) diet induced atherosclerosis compared to age and sex matched controls. To investigate potential mechanisms that may explain this paradoxical dissociation between plasma concentrations of HDL and atherosclerosis we have used recombinant adenovirus vectors to express, in vivo, two other proteins that play a major role in HDL metabolism. Adenovirus-mediated expression of HL and CETP in both control and L-tg mice demonstrated that compared to the HDL in control animals, the apoA-I/A-II HDL in L-tg mice were poorly hydrolyzed by HL and would not support the transport of cholesteryl esters to apoB containing lipoproteins by CETP. Additionally, baseline plasma concentrations of nascent, pre-B1-HDL, the most effector particles for the efflux of cellular cholesterol, were significantly reduced in L-tg mice. The decay plasma curve of L-tg mouse HDL after injection of radiolabelled HDL-cholesterol ether isolated from control and L-tg mice was significantly delayed from that of control mouse HDL. In addition, the % of total cholesterol ether delivered to the liver, two hours after injection of radiolabelled HDL, was significantly reduced (P<0.001) for L-tg mice compared to controls (18% versus 33% of total cholesterol ether). These combined studies indicate that in the absence of CETP, LCAT overexpression in mice leads to the formation of HDL particles with abnormal composition and function, establishing one potential mechanism that may account for the enhanced atherosclerosis observed in this animal model. Thus, the process of reverse cholesterol transport, an important mechanism by which HDL may modulate the development of atherosclerosis is interrupted in L-tg mice. These studies demonstrate that HDL and apoA-I plasma concentrations, alone, are not predictive of the anti-atherogenic potential of HDL and functional studies are required to determine whether increasing plasma HDL by different therapeutic modalities will if fact, protect against the development of premature cardiovascular disease.